Distribution Of Three Intestinal Protists In Hospital Wastewater And The Zoonotic Characteristics Of Cryptosporidium Felis And Cryptosporidium Canis

Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi are three important pathogens of diarrhea.The infection of these intestinal protists by fecal-oral route and waterborne transmission has caused significant public concerns.Although hospitals are potential sources of these intestinal protists,their distribution in hospital wastewater remains unclear.Among Cryptosporidium spp.,Cryptosporidium felis and Cryptosporidium canis are two of five major human-pathogenic species,which could infect both companion animals and humans.Due to the lack of genomic data and subtyping tools,the genetic diversity,host and geographical distribution of C.felis and C.canis are still poorly understood,their potential zoonotic transmission route also needs to be confirmed.Thus,here we carried out the following study:To investigate the distribution of Cryptosporidium spp.,G.duodenalis and E.bieneusi in hospital wastewater,wastewater samples were collected from two hospitals and a downstream wastewater treatment plant in Shanghai,China.Cryptosporidium spp.,G.duodenalis and E.bieneusi were detected and identified using PCR and DNA sequencing techniques.Among 164 samples analyzed,31(18.9%),45(27.4%),and 122(74.4%)were positive for these pathogens,respectively.Cryptosporidium hominis,G.duodenalis assemblage A,and E.bieneusi genotype D were the dominant species,assemblage and genotype,respectively.The distribution characteristics of these intestinal protists are similar between samples from hospitals and the wastewater treatment plant,which indicates that hospitals are one of the potential contributors of these pathogens in urban waters.Moreover,the detected G.duodenalis assemblage A and E.bieneusi genotype D have also been commonly found in local companion animals,which suggests that companion animals might also play roles in the transmission of these pathogens.To genetically analyze the zoonotic characteristics of C.felis and C.canis,oocysts from 31 C.felis-positive and C.canis-positive specimens were purified by utilizing immunomagnetic isolation.After whole genome amplification and real time PCR purity-verification,genomic sequences of two C.felis isolates and one C.canis isolate were obtained by using Illumina HiSeq technique.By contrast to the published Cryptosporidium genomes,the obtained genomes differed significantly in GC contents(39.7-40.1%versus 24.3-33.0%),which has revealed the genetic uniqueness of C.felis and C.canis.Based on the obtained C.felis genome,a subtyping tool targeting the C.felis 60 kDa glycoprotein(gp60)gene was developed.Combined with the published gp60-based subtyping tool,66 C.felis isolates derived from humans and animals worldwide were analyzed in this study.Based on the sequence polymorphism and phylogenetic analyses of partial gp60 gene,44 obtained sequences formed 23 subtypes clustered in four distinct subtype families,namely??a,??c,??d and ??e.Among them,the dominant subtype family ??a had zoonotic feature and different geographic distribution.In contrast,all ??d isolates derived from humans from various countries.A subtyping tool targeting the C.canis gp60 gene was developed for the first time in this study.To investigate the genetic diversity and epidemiologic characteristics of C.canis,a total of 76 C.canis isolates from various hosts and countries were subtyped by using the developed tool.Phylogenetically,49 obtained gp60 sequences generated ten subtypes clustered in five distinct subtype families,namely XXa-XXe.Among them,the dominant subtype family XXa could infect both humans and domestic dogs.In addition,the occurrence of subtype XXa4 in two children and a dog in a Peruvian household has further confirmed the zoonotic characteristics of C.canis.In conclusion,we analyzed the distribution of intestinal protists in hospital wastewater,sequenced the genomes of C felis and C.canis for the first time,complemented the subtyping data for C.felis,and developed a subtyping tool for C.canis.Subtyping data from various hosts worldwide have revealed the epidemiologic features at the subtype level of C.felis and C.canis.Findings of this study might be helpful to better understand the distribution characteristics and transmission dynamics of the involved pathogens and to provide a theoretical guidance to the formulation of their preventive measures.